The present invention relates to compositions obtainable from plants, and methods for enhancing disease resistance of plants using the compositions. More particularly, the invention relates to nucleic acid compositions and encoded polypeptides, as well as microorganisms and plants transformed with the nucleic acid for production of encoded polypeptides.
When a plant pathogen interacts with a potential host, it may successfully colonize the host and cause disease, in which case the pathogen is said to be virulent, the host is susceptible, and the interaction is compatible. Alternatively, the plant may respond to the pathogen by rapidly activating a battery of defense responses, interfering with pathogen multiplication and preventing disease. In this case, the pathogen is said to be avirulent, the host is resistant, and the interaction is incompatible. The outcomes of plant-pathogen interactions often fit a xe2x80x9cgene-for-genexe2x80x9d model. In this model, resistance results when the pathogen carries a particular avirulence gene that corresponds to a particular resistance gene (R gene) in the host. In general, each R gene confers resistance only to pathogens carrying the corresponding avirulence gene. Gene-for-gene resistance responses have been observed in interactions of plants with a wide variety of pathogens, including fungi, bacteria and viruses. A simple molecular explanation for gene-for-gene resistance is that avirulence genes encode ligands that bind to receptors encoded by the plant R genes or that avirulence gene products synthesize such ligands. Ligand binding triggers activation of a signal transduction cascade culminating in expression of defense responses that inhibit the pathogen and confer resistance. The hallmark of gene-for-gene resistance is a rapid programmed cell death of the cells in contact with the pathogen, called the hypersensitive response, or HR.
In many cases, gene-for-gene resistance reactions trigger another form of strong disease resistance called systemic acquired resistance, or SAR. Infection by a necrotizing pathogen (a pathogen that causes host cell death) causes a signal to be transmitted throughout the plant. In response, defense genes are activated in uninfected tissue, and the plant shows resistance to subsequent infection by a wide range of normally compatible pathogens. It is clear that salicylic acid (SA) plays a key role in establishment of SAR; resistant tissue contains elevated levels of SA, treatment of plants with SA induces defense gene expression and resistance, and SA is required in the responding tissue for defense gene expression and resistance. The question of whether or not SA is also the systemic signal has not yet been settled.
Genetic analyses using Arabidopsis-pathogen systems are being used to dissect the signaling pathways governing gene-for-gene resistance and SAR. Common pathogens used include the virulent Pseudomonas syringae strains Pseudomonas syringae pv. maculicola (Psm ES4326) and Pseudomonas syringae pv. tomato (Pst DC3000), Gram-negative bacteria that cause xe2x80x9cbacterial speckxe2x80x9d diseases in many crop plants, as well as isogenic strains carrying any of several cloned avirulence genes that elicit gene-for-gene resistance responses. In addition, a large number of isolates of Peronospora parasitica have been characterized and used to define many R genes in various Arabidopsis accessions. Several R genes have been isolated from Arabidopsis and other species. Comparison of the amino acid sequences of the R proteins has resulted in their division into several major classes.
Less progress has been made in identifying factors acting immediately downstream of R genes in gene-for-gene resistance. The Arabidopsis mutants ndr1 and eds1 have properties suggesting that NDR1 and EDS1 may be such factors. Mutations in either ndr1 or eds1 interfere with gene-for-gene resistance conferred by subsets of R genes.
Infection of Arabidopsis by P. syringae induces many defense responses, including synthesis of the phytoalexin camalexin, and expression of the pathogenesis-related genes PR-1 and PR-5, xcex2-glucanase (BGL2), and anthranilate synthase (ASA1). Phytoalexins are small molecule broad-spectrum antimicrobial compounds synthesized by plants in response to pathogen attack. Camalexin is the only phytoalexin produced in significant quantities by Arabidopsis. Infection by a P. syringae strain carrying an avirulence gene such as avrRpt2 or treatment with SA induces SAR. SAR correlates with systemic expression of PR-1, PR-5, and BGL2.
SA-dependent signaling has been studied using genetic analysis in Arabidopsis. The central role of SA in defense response signaling was revealed by characterization of transgenic plants expressing nahG. The nahG transgene encodes salicylate hydroxylase, an enzyme that converts SA to catechol. Thus, nahG plants are unable to accumulate SA, and are conceptually similar to mutants deficient in SA. Arabidopsis nahG plants fail to develop SAR in response to SA or necrotizing pathogens. They are also compromised in local resistance, displaying reduced PR-1 expression in response to infection, susceptibility to pathogens that are normally avirulent as a consequence of gene-for-gene resistance, and heightened susceptibility to normally virulent pathogens. Although camalexin synthesis is not inducible by SA, nahG plants fail to synthesize camalexin in response to local Psm ES4326 infection, suggesting that SA is necessary, but not sufficient, for activation of camalexin synthesis. Taken together, these results show that SA is important in gene-for-gene resistance and in limiting growth of virulent pathogens, as well as in SAR.
Several Arabidopsis mutants that constitutively express SAR have been isolated. Many of these mutants are xe2x80x9clesion-mimicsxe2x80x9d, that is, they spontaneously develop necrotic lesions in the absence of any pathogen. After developing lesions, these plants accumulate high levels of SA, express PR-1 and are more resistant to pathogens. It is thought that lesion formation mimics the HR, thereby activating the SAR pathway in the rest of the plant. The lesion-mimic mutations appear to be acting upstream from SA in that they exhibit elevated SA levels and introduction of nahG reduces SA levels and abolishes PR-1 expression and resistance. In some, but not all lesion-mimics, introduction of nahG also abolishes lesion formation, strongly suggesting the existence of a positive feedback loop between cell death and SA accumulation. The cpr1 and cpr6 mutants constitutively express PR-1 and exhibit elevated SA levels, but do not spontaneously develop lesions. These mutations may define genes acting between lesion formation and SA accumulation. Importantly, lesion mimic mutants generally have greatly reduced vigor, and cpr1, cpr5, and cpr6 plants are dwarf, indicating that constitutive expression of defense responses may not be the best strategy for improving disease resistance in crops.
An Arabidopsis gene that has been variously named NPR1, NIM1, and SAI1, is required for SA-mediated disease resistance. SA treatment of npr1/nim1 /sai1 mutants does not activate expression of PR-1, PR-5, or BGL2. When infected with a pathogen that induces SAR in wild-type plants, npr1/nim1/sai1 mutants accumulate high levels of SA, but do not develop SAR, indicating that NPR1/NIM1/SAI1 acts downstream from SA. However, NPR1/NIM1/SAI1 is not required for camalexin synthesis, so the effect of SA on camalexin synthesis must be mediated by some other, as yet unknown, factor. Plants with npr1/nim1/sai1 mutations display enhanced susceptibility to virulent pathogens, demonstrating that signal transduction downstream from NPR1/NIM1/SAI1 is important for restricting virulent pathogens as well as for SAR. NPR1/NIM1/SAI1 was recently cloned and shown to encode a protein containing ankyrin repeats.
Improving plant germplasm for increased resistance to disease is an agronomic goal of a high priority. Plants better able to respond to disease challenge in general, and via timely SA-induced phytoalexin synthesis in particular, are desirable.
A problem with the prior art is that genes available for use in genetic transformation to improve regulation of SA-mediated defense responses in plants are not numerous. As a result, efforts to improve plant germplasm for increased resistance to disease have been hampered. A challenge for genetic engineering has been to develop plants that can express disease defense responses without deleterious side effects. The present invention seeks to overcome this and other drawbacks inherent in the prior art.
ABRC: Arabidopsis Biological Resource Center
ASA1: protein encoded by anthranilate synthase gene ASA1
ATCC: American Type Culture Collection
BAC: bacterial artificial chromosome
BGL2: protein encoded by xcex2-glucanase gene BGL2
CAPS: cleaved amplified polymorphic sequence
CSPD Disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2xe2x80x2-(5xe2x80x2-chloro)tricyclo[3.3.1.13.7]decan}-4-yl)phenyl phosphate
Col: Columbia ecotype
HR: hypersensitive response
JA: jasmonic acid
Ksk: Keswick ecotype
Ler: Landsberg erecta ecotype
NMD: nonsense mediated mRNA decay
PAD4: gene encoding wild-type PAD4 protein
PAD4: wild-type protein
pad4-1: gene encoding mutant pad4-1 protein
pad4-2: gene encoding mutant pad4-2 protein
pad4-3: gene encoding mutant pad4-3 protein
pad4-4: gene encoding mutant pad4-4 protein
PCR: polymerase chain reaction
PR-1: protein encoded by pathogenesis-related gene PR-1
PR-5: protein encoded by pathogenesis-related gene PR-5
Psm: Pseudomonas syringae pv maculicola 
Pst: Pseudomonas syringae pv tomato 
RACE: Random Amplified cDNA End
RFLP: Restriction Fragment Length Polymorphism
SA: salicylic acid
SAG: salicylic acid glucoside
SAR: systemic acquired resistance
YAC: yeast artificial chromosome
The present invention provides PAD4 compositions, and methods for using PAD4 compositions for enhancing disease resistance of a plant. PAD4 plays an important role in disease resistance since pad4 mutants show enhanced susceptibility to pathogen attack. Pathogens that show more extensive growth on pad4 plants than on wild-type plants include virulent strains of the bacterial pathogen Pseudomonas syringae, and avirulent and virulent isolates of the oomycete pathogen Peronospora parasitica, for example. Characterization of pad4 mutants, as provided herein, demonstrated that PAD4 acts by activating expression of defense mechanisms in response to pathogen attack. Additional analysis of SA and pathogen-induced PAD4 expression in pad4 mutants, also as provided herein, evidenced that PAD4 and SA act in a positive signal amplification loop required for activation of defense responses. The present inventors determined the nucleotide sequence of over 11,000 bp of DNA adjacent to and including the gene encoding PAD4, and provide herein nucleic acid and protein sequences for compositions for improving disease resistance in plants.
Table 1 of the Detailed Description infra provides identification of sequences having sequence identifiers for the present invention.
In particular, the invention provides a purified nucleic acid molecule comprising a sequence of nucleotides encoding a PAD4 polypeptide, the PAD4 polypeptide having an amino acid sequence essentially as set forth in SEQ ID NO. 2, or SEQ ID NO. 55.
In a further embodiment, the invention provides a purified nucleic acid molecule having a nucleotide sequence selected from the group consisting of:
a) the nucleotide sequence essentially set forth as SEQ ID NO.:1 or SEQ ID NO.:54;
b) the nucleotide sequence essentially set forth as SEQ ID NO.:3, SEQ ID NO.: 56, SEQ ID NO.: 58, SEQ ID NO.:60, or SEQ ID NO.:62;
c) the nucleotide sequence essentially set forth as SEQ ID NO.:5;
d) a nucleotide sequence encoding part or all of a polypeptide having an amino acid sequence essentially set forth as SEQ ID NO.: 2 or SEQ ID NO.: 55;
e) a nucleotide sequence that, through degeneracy of the genetic code, encodes part or all of essentially the same polypeptide as that encoded by the nucleotide sequence of any of a)-d);
f) a nucleotide sequence that is the complement of any of a)-e); and
g) a nucleotide sequence that hybridizes to any of a)-f) under conditions of predetermined stringency.
Another embodiment of the invention is an oligonucleotide molecule comprising a nucleotide sequence between 20 and 100 nucleotides in length, the molecule hybridizing under conditions of predetermined stringency with a portion of an above-named nucleic acid molecule.
Nucleic acid molecules of the invention are derived from any plant species, including, without limitation, angiosperms (for example, dicots and monocots) and gymnosperms. Exemplary plants from which the nucleic acid may be derived, and into which the nucleic acid may be introduced as a transgene, include, for example, plants such as sugar cane, wheat, rice, maize, sugar beet, potato, barley, manioc, sweet potato, soybean, sorghum, cassava, grape, oats, millet, rye, watermelon, canola, garlic, strawberry, bean, mango, alfalfa, apple, banana, coffee, oil seed Brassica, coconut, cotton, sunflower, olive, papaya, peanut, safflower, sesame, flax, palm, arugula, asparagus, artichoke, edible beans, beet, broccoli, brussel sprouts, cabbage, carrot, cauliflower, celery, cilantro, cucumber, eggplant, endive, horseradish, lettuce, okra, onion, parsnip, pea, hot pepper, sweet pepper, pumpkin, radish, spinach, squash, sweet corn, sweet potato, swiss chard, tomato, turnip, yam, or zucchini. Preferred nucleic acid molecules are derived from cruciferous plants, for example, Arabidopsis thaliana. An example of such a cruciferous nucleic acid molecule is provided as SEQ ID NO.:1, SEQ ID NO.:3, SEQ ID NO.:54, SEQ ID NO.: 56, SEQ ID NO.: 58, SEQ ID NO.:60, or SEQ ID NO.:62, or SEQ ID NO.:5. In a preferred embodiment, the purified nucleic acid molecule as described herein is from Arabidopsis.
The invention also provides a purified protein comprising a sequence of amino acids, the protein having positive regulatory effect on phytoalexin levels and on PR-1 expression levels while having substantially no regulatory effect on PR-5, BGL2, or ASA1 expression levels in a disease defense response by a host plant. The invention further provides a purified protein comprising a sequence of amino acids, the protein having positive regulatory effect on its own expression levels, on phytoalexin levels, and on PR-1 expression levels while having substantially no regulatory effect on PR-5, BGL2, or ASA1 expression levels in a disease defense response by a host plant. Such protein may be obtained from any plant species, for example, those plants listed herein. In preferred embodiments, the protein is derived from a cruciferous species, for example, Arabidopsis thaliana, or from a solanaceous species, for example, Nicotiana glutinosa. In a further preferred embodiment, the protein comprises an amino acid sequence essentially as set forth in SEQ ID NO.: 2 or SEQ ID NO.: 55. A purified protein produced by expression of an above-described nucleic acid molecule in an expression vector is a further aspect of the invention.
In a related embodiment, the invention further provides a recombinant vector comprising a purified nucleic acid molecule as described herein. The recombinant vector may be an expression vector comprising a regulatory element for expression of PAD4 in plant cells. In a preferred embodiment, the purified nucleic acid molecule of the invention is operably linked to an expression control region that mediates expression of a polypeptide encoded by the nucleic acid molecule. For example, the expression control region is capable of mediating constitutive, inducible (for example, pathogen- or wound-inducible), or cell- or tissue-specific gene expression.
A recombinant host cell comprising a nucleic acid molecule as described herein and a recombinant host cell comprising a recombinant vector as described herein are also aspects of the present invention. In preferred embodiments, the recombinant host cell is a bacterium (for example, E. Coli or Agrobacterium tumefaciens) or is a plant cell (for example, a cell from any of the plants listed herein). Such a plant cell transformed with and expressing a gene encoding PAD4 has an increased level of resistance against disease, in particular, a disease caused by a plant pathogen (for example, Phytophthora, Peronospora, or Pseudonomas). In one embodiment, the gene encoding PAD4 is heterologous to the plant cell. Preferably, the heterologous gene is integrated into the plant genome.
In another embodiment, the invention provides a plant comprising a transgene expressing PAD4, thereby providing enhanced disease resistance to the plant. Preferably, the transgene is integrated into the genome of the plant, wherein the nucleic acid molecule is expressed in the transgenic plant. The transgene may be heterologous or homologous. In addition, the invention provides progeny, seeds, or clones of such transgenic plants. Such transgenic plants may be produced according to conventional methods using any of the plants listed supra.
A purified antibody having binding specificity for an antigenic region of PAD4 or PAD4 is a further aspect of the invention.
In another aspect, the invention provides a method of using a nucleic acid molecule as provided herein, the method comprising: preparing a recombinant vector in which the nucleic acid molecule is positioned under the control of a promoter; introducing the recombinant vector into a host cell; culturing the host cell under conditions effective to allow expression of the encoded polypeptide; and collecting the expressed polypeptide. A recombinant protein produced by such a method is a further aspect of the present invention.
A method for enhancing resistance of a plant to disease comprising providing to the plant an amount of PAD4 effective to enhance resistance to disease is provided herein. The method may comprise producing a transgenic plant cell including a nucleic acid molecule encoding PAD4, wherein the nucleic acid molecule is positioned for expression in the plant cell; and growing a transgenic plant from the plant cell; wherein the nucleic acid molecule is expressed in the transgenic plant to produce PAD4, thereby providing an increased level of resistance to disease. The disease may be due to a pathogen selected from the group consisting of a bacterium, virus, viroid, fungus, oomycete, nematode, or insect. In particular, the disease may be due to infection by Phytophthora, Peronospora, or Pseudomonas.
The present invention provides a method of determining presence of target nucleic acid in a test plant cell, comprising: contacting a nucleic acid molecule as provided herein, or a portion thereof, with a preparation of nucleic acid from the test plant cell under hybridization conditions of predetermined stringency; and assessing hybridization of the nucleic acid molecule in the preparation, wherein when hybridization occurs under conditions of predetermined stringency, target nucleic acid is present in the test plant cell. The method may further comprise the step of isolating the target nucleic acid.
The present invention provides a further method of determining presence of target nucleic acid in a test plant cell comprising: providing a sample of test plant cell nucleic acid; contacting at least one oligonucleotide having sequence identity to a region of the nucleic acid of SEQ ID NO.:1 or SEQ ID NO.:54 with the test plant cell nucleic acid under conditions suitable for nucleic acid amplification; amplifying the nucleic acid; and determining presence of target nucleic acid by presence of amplified nucleic acid. The method may further comprise the step of isolating the amplified target nucleic acid.
A method of producing a transgenic plant, comprising incorporating a nucleic acid molecule as described herein into a plant cell and generating a transgenic plant from the plant cell is a further embodiment of the invention.
A further aspect of the present invention is a method for enhancing resistance of a plant to disease comprising providing to the plant an amount of a nucleic acid molecule effective to enhance resistance to disease, the nucleic acid molecule comprising a sequence of nucleotides encoding a factor, the factor having a positive regulatory effect on phytoalexin levels and on PR-1 expression levels while having substantially no regulatory effect on PR-5, BGL2, or ASA1 expression levels in a disease defense response by a host plant. An additional aspect of the present invention is a method for enhancing resistance of a plant to disease comprising providing to the plant an amount of a nucleic acid molecule effective to enhance resistance to disease, the nucleic acid molecule comprising a sequence of nucleotides encoding a factor, the factor having a positive regulatory effect on its own expression levels, on phytoalexin levels, and on PR-1 expression levels while having substantially no regulatory effect on PR-5, BGL2, or ASA1 expression levels in a disease defense response by a host plant. In a preferred embodiment, the factor is PAD4. In a further preferred embodiment, the nucleic acid molecule has a nucleotide sequence essentially as set forth as SEQ ID NO.:5. In another further preferred embodiment, the purified protein comprising a sequence of amino acids having a positive regulatory effect on phytoalexin levels and on PR-1 expression levels while having substantially no regulatory effect on PR-5, BGL2, or ASA1 expression levels in a disease defense response by a host plant is an esterase or a lipase.
The major features of the plant defense response that have been observed in crop plants have also been observed in Arabidopsis-pathogen interactions. For example, several resistance gene-avirulence gene interactions have been identified for both bacterial and fungal pathogens of Arabidopsis. Further, the important features of systemic acquired resistance have been observed in Arabidopsis. Arabidopsis genetic analysis has been used to help identify a variety of components of the Arabidopsis defense response to pathogen attack. Thus, the invention as provided herein provides the basis for identifying PAD4 genes that are involved in disease resistance throughout the plant kingdom and are not limited to Arabidopsis.
PAD4 compositions of the present invention provide a number of important advances and advantages for the protection of plants against their pathogens and against environmental stress. For example, by providing PAD4 genes as described herein that are readily incorporated and expressed in all species of plants, the invention facilitates an effective and economical means for in-plant protection against plant disease. Such protection against disease reduces or minimizes the need for traditional chemical practices (for example, application of fungicides, bactericides, nematicides, insecticides, or viricides) that are typically used by farmers for controlling the spread of plant pathogens and providing protection against disease. In addition, because plants expressing a PAD4 gene described herein are less vulnerable to pathogens and their diseases, the invention further provides for increased production efficiency, as well as for improvements in quality and yield of crop plants and ornamentals. Thus, the invention contributes to the production of high quality and high yield agricultural products: for example, fruits, ornamentals, vegetables, cereals and field crops having reduced spots, blemishes, and blotches that are caused by pathogens; agricultural products with increased shelf-life and reduced handling costs; and high quality and yield crops for agricultural (for example, cereal and field crops), industrial (for example, oilseeds), and commercial (for example, fiber crops) purposes. Furthermore, because the invention reduces the necessity for chemical protection against plant pathogens, the invention benefits the environment where the crops are grown. Genetically-improved seeds and other plant products that are produced using plants expressing the genes described herein also render farming possible in areas previously unsuitable for agricultural production.
The invention is also useful for providing nucleic acid and amino acid sequences of a PAD4 gene that facilitates the isolation and identification of PAD4 genes, as well as the isolation and identification of genes related to PAD4 genes (such as mutant alleles or PAD4-like pseudogenes), from any plant species. In particular, all or a portion of nucleic acid molecules having the nucleotide sequence essentially set forth as SEQ ID NO.:1, SEQ ID NO.:3, SEQ ID NO.: 54, SEQ ID NO.:56, SEQ ID NO.:58, SEQ ID NO.:60, or SEQ ID NO.:62, or having a nucleotide sequence complementary to such a nucleotide sequence, may be used as probes for the isolation and identification of PAD4 genes, as well as the isolation and identification of genes related to PAD4 genes (such as mutant alleles or PAD4-like pseudogenes), from any plant species. The nucleic acid and protein molecules of the present invention have further utility as molecular weight markers for separation procedures using gels and chromatography columns, for example; and fragments of nucleic acids are useful as probes and primers, for example, especially those having greater than 50% G+C content.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
Following long-standing patent law convention, the terms xe2x80x9caxe2x80x9d and xe2x80x9canxe2x80x9d mean xe2x80x9cone or morexe2x80x9d when used in this application, including the claims. Even though the invention has been described with a certain degree of particularity, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing disclosure. Accordingly, it is intended that all such alternatives, modifications, and variations which fall within the spirit and the scope of the invention be embraced by the defined claims.